Role of protein kinase M zeta in spinal nociceptive sensitization

重复或强烈的伤害性刺激导致外周神经和中枢神经系统中的神经元（包括脊髓背角（SCDH））的敏化。虽然各种谷氨酸受体和下游细胞内信使有助于启动SCDH的伤害性敏化，脊髓伤害性敏化的维持知之甚少。蛋白激酶M zeta（PKMzeta）是一种持续活性的激酶，其独特地有助于维持海马中的长时程增强（LTP）和记忆储存，并且可能有助于脊髓伤害性敏化。我们已经表明，PKMzeta在SCDH中响应于持续的伤害性刺激或脊髓神经元的直接激活而上调，并且PKMzeta抑制剂逆转降低的伤害性阈值（即，异常性疼痛），包括由足底内（i.pl.）辣椒素或鞘内（i.t.）DHPG，或肌肉内（i. m.）在大鼠大腿中注射酸性盐水。这三种刺激还诱导脊髓宽动态范围神经元对后爪感受野的机械刺激的敏感性，这被PKMzeta的抑制剂逆转。 尽管已知各种蛋白激酶（PKs）（如PKA、PKC、Ca 2 +/钙调蛋白依赖性PK-II（CaMK-II）、丝裂原相关PK（MAPK）和磷脂酰肌醇3-激酶（PI 3-激酶））引发脊髓伤害性敏化，和其他介质，如与NIMA 1（PIN-1）和磷酸肌醇依赖性PK-1（PDK 1）相互作用的肽基脯氨酰异构酶蛋白尽管这些介质调节海马中的PKMzeta活性，但在脊髓伤害性敏化期间这些介质中是否有任何调节PKMzeta活性是未知的。使用选择性PK抑制剂和PKMzeta和p-PKMzeta的蛋白质测定，我们建议确定哪些介质有助于外周和脊髓伤害性刺激诱导的PKMzeta的长期上调和激活。此外，已经表明PKMzeta通过增加海马神经元中突触后AMPA受体的水平来维持记忆，并且持续的伤害感受触发SCDH AMPA受体的亚细胞分布的改变。因此，为了评估脊髓中有害刺激诱导的PKMzeta激活的可能后果，我们将使用亚细胞分级分离和蛋白质测定来检查PKMzeta对SCDH神经元中AMPA受体亚细胞分布改变的贡献。最后，最近的研究质疑zeta抑制肽的特异性（迄今为止在脊髓伤害性敏化研究中使用的唯一抑制剂），以及显示在PKCzeta/PKMzeta敲除小鼠中LTP和记忆处理不受影响。因此，我们建议确定PKMzeta基因操作的影响，使用显性负PKMzeta慢病毒载体在大鼠或标准和条件性基因敲除小鼠，对维持异常性疼痛和/或致敏的背角神经元诱导外周或脊髓伤害性刺激。 这些研究将带来新的见解和方法，应有助于理解脊髓伤害性敏化的基础神经可塑性过程。了解这些过程并开发治疗方法来逆转或消除有害刺激对神经系统（特别是脊髓神经元）的影响，将比开发消除记忆的方法更实用，并且具有更大的影响。

Repeated or intense noxious stimulation leads to a sensitization of both peripheral nerves and neurons in the central nervous system, including the spinal cord dorsal horn (SCDH). Although various glutamate receptors and downstream intracellular messengers contribute to the initiation of nociceptive sensitization in SCDH, little is known about the maintenance of spinal nociceptive sensitization. Protein kinase M zeta (PKMzeta) is a persistently active kinase that contributes uniquely to the maintenance of long-term potentiation (LTP) and memory storage in the hippocampus, and may contribute to spinal nociceptive sensitization. We have shown that PKMzeta is upregulated in SCDH in response to persistent noxious stimulation or direct activation of spinal neurons, and that a PKMzeta inhibitor reverses lowered nociceptive thresholds (i.e., allodynia) that depend on spinal neuroplasticity, including allodynia induced by intraplantar (i.pl.) capsaicin or intrathecal (i.t.) DHPG, or referred hind paw allodynia after intramuscular (i.m.) injections of acid saline in the rat thigh. These three stimuli also induced a sensitization of spinal wide dynamic range neurons to mechanical stimulation of their hind paw receptive fields, which was reversed by an inhibitor of PKMzeta. Although it is known that various protein kinases (PKs) (such as PKA, PKC, Ca2+/calmodulin-dependent PK-II (CaMK-II), mitogen-associated PK (MAPK) and phosphatidylinositol 3-kinase (PI3-kinase)) initiate spinal nociceptive sensitization, and other mediators such as peptidyl-prolyl isomerase protein interacting with NIMA 1 (PIN-1) and phosphoinositide-dependent PK-1 (PDK1) regulate PKMzeta activity in hippocampus, it is unknown whether any of these mediators regulate PKMzeta activity in during spinal nociceptive sensitization. Using selective PK inhibitors and protein assays of PKMzeta and p-PKMzeta, we propose to determine which of these mediators contributes to the prolonged upregulation and activation of PKMzeta induced by peripheral and spinal nociceptive stimulation. Also, it has been shown that PKMzeta maintains memory by increasing the levels of post-synaptic AMPA receptors in hippocampal neurons, and that persistent nociception triggers alterations in the subcellular distribution of SCDH AMPA receptors. Thus, to assess possible consequences of noxious stimulus-induced PKMzeta activation in spinal cord, we will use subcellular fractionation and protein assays to examine the contribution of PKMzeta to alterations in the subcellular distribution of AMPA receptors in SCDH neurons. Lastly, recent studies have questioned the specificity of zeta-inhibitory peptide (the only inhibitor used thus far in studies of spinal nociceptive sensitization), as well as showing that LTP and memory processing are unaffected in PKCzeta/PKMzeta knockout mice. Therefore, we propose to determine the effects of gene manipulation of PKMzeta, using a dominant-negative PKMzeta lentiviral vector in rats or standard and conditional knock-out mice, on the maintenance of allodynia and/or sensitization of dorsal horn neurons induced by peripheral or spinal nociceptive stimulation. These studies will bring new insights and approaches that should aid in understanding the fundamental neuroplastic processes that underlie spinal nociceptive sensitization. Understanding the processes and developing treatments to reverse or erase the effects of noxious stimuli on the nervous system (particularly spinal neurons) will be more practical, and have considerably more impact, than developing ways to erase memories.